The matrix metalloproteinase (MMP) family consists of at least 23 structurally related, soluble or membrane bound zinc-dependent endopeptidases that are broadly involved in the remodelling of the extracellular matrix (ECM) and in the functional regulation of various bioactive molecules.
All MMPs possess a prototype structure that includes a pro-domain that maintains the MMP in an inactive form and a catalytic domain that acts on a broad spectrum of extracellular matrix components.
Matrix metallopeptidase 9 (MMP9), also known as 92 kDa type IV collagenase or gelatinase B (GELB), is a member of the MMP family enzymes responsible for the degradation of denatured and basement membrane collagens (Agrawal et al., 2006 J. Exp. Med. 203, 1007-1019) and for promoting inflammation by processing of soluble proteins, including protease inhibitors (Liu et al., 2000, J. Exp. Med. 188, 475-482.), chemokines (Van den Steen et al., 2000, Lancet Neurol. 2, 747-756), and cytokines (Nelissen et al., 2003, Brain 126, 1371-1381). MMP9 also controls migration, invasion and metastasis of tumor cells by proteolysis of membrane-bound molecules, like growth factor precursors and receptors, tyrosine kinase receptors (TKRs), cell adhesion molecules (Bauvois, 2012, Biochim Biophys Acta. 1825(1):29-36.). In disease MMP9 is secreted by many cell types including leukocytes e.g. neutrophils, monocytes/macrophages, and lymphocytes, as well as fibroblasts, myofibroblasts, epithelial cells, smooth muscle cells, endothelial cells, osteoclasts and tumor cells (Vandooren et al., 2013, Crit. Rev. Biochem. Mol. Biol. 48(3):222-72).
The general domain structure of MMP9 comprises a secretory leader sequence, an inhibitory pro-domain required for catalytic latency, a ‘split’ catalytic domain containing three fibronectin type II-like repeat loops that together form a collagen binding domain (CBD), a hyperglycosylated proline-rich linker (also referred to as the OG domain), and a hemopexin-like C-terminal domain (PEX).
Matrix metallopeptidase 2 (MMP2), also known as 72 kDa type IV collagenase or gelatinase A (GELA), is an enzyme that belongs to the same family as MMP9. MMP2 and MMP9 exhibit high amino acid sequence identity (45.9% on full length protein and 63.2% on catalytic domain) and share a highly similar 3D-structure, especially in their catalytic domain. It is therefore very difficult to identify inhibitory anti-human MMP9 antibodies selective versus human MMP2 due to this high structural and amino acid sequence homology (Morgunova et al., 1999, Science, 284: 1667-1670).
Many acute inflammatory and autoimmune disease states, fibrotic conditions and invasive cancer, are associated with the presence of excessive MMP9 (Hu et al, 2007, Nature Reviews Drug Discovery, 6, 480-498; Ram et al, 2006, J. Clin. Immunol., (26)4: 299-307; Ai Zheng, 2003, Chinese journal of cancer, 22(2):178-84; Baugh et al, 1999, Gastroenterology, 117:814-822; Santos et al, 2013, Biochem Biophys Res Commun., 438(4): 760-4); Herszényi et al, 2012, Int. J. Mol. Sci., 13, 13240-13263; Lijnen, 2001, Thromb Haemost, 86: 324-33; Rosell et al., 2005, Stroke 36: 1415-20; Whatling et al., 2004, Arterioscler Thromb Vasc Biol., 24: 10-11; Yasmin et al., 2005, Arterioscler Thromb Vasc Biol., 25:372-8; Vassiliadis et al., 2011, BMC Dermatol., 11: 6) and, thus, this enzyme has received considerable attention as a prospective target for therapeutic intervention.
Strong clinical and experimental evidence demonstrates association of elevated levels of MMP9 with cancer progression, metastasis and shortened patient survival, as it plays a key role in tumor cell invasion and metastasis by digesting the basement membrane and extra cellular matrix components. Neutrophil gelatinase-associated lipocalin (NGAL), which is covalently linked to MMP9 in human neutrophils (Triebel et al., 1992, FEBS Lett., 314, 386-388), protects MMP9 from proteolytic degradation and increases the enzymatic activity of MMP9 and subsequently enhances tumoral invasiveness and diffusion (Yan et al., 2001, J. Biol. Chem., 276, 37258-37265). High concentrations of MMP9/NGAL complex in serum have been associated with a shorter progression-free survival and poor overall survival in clear cell renal cell carcinoma (Perrin et al., 2011, Prog. En Urol. J. Assoc. Fr. Urol. Société Fr. Urol., 21, 851-858).
Specifically the role of MMP-9 has been associated with colorectal cancer (Herszényi et al., 2012, Int J Mol Sci., 13(10):13240-63), pancreatic cancer (Gao et al., 2015, Med Oncol. 32(1): 418), breast cancer (Kim et al., 2014, BMC Cancer. 14(1):959), lung cancer (Ruiz-Morales et al., Tumour Biol. 36(5):3601-10),), ovarian cancer (Naylor et al., 1994, Int J Cancer, 58: 50-6), urinary bladder cancer (Szarvas et al., 2011, Nat Rev Urol., 8(5):241-54) and gastric cancer (Chen et al., 2015, Int J Clin Exp Med. 8(1):546-57).
The role of MMP9 has been shown in immune pathologies and particularly in inflammatory bowel disease (IBD) where MMP9, is reported as the most abundantly expressed MMP in actively inflamed bowel mucosa, and its expression correlates well with disease activity (Naito and Yoshikawa, 2005, 26:379-390). In IBD, MMP9 is thought to play a key role in inadequate tissue remodeling and activation of proinflammatory cytokines and chemokines thereby enabling the recruitment of activated leukocytes (Nuala et al., 2014, Inflamm. Bowel 0:1-15). More specifically, enhanced MMP9 expression along fistula tracts of perianal fistulae and increased MMP9 activity in fistula biopsies was reported in Crohn's disease (CD) patients, supporting the hypothesis that MMP9 may contribute to the formation of fistula that represent a severe complication of CD (Efsen, et al., 2011, Basic Clin Pharmacol Toxicol. 109(3):208-16). Furthermore, decreased NGAL/MMP9 serum levels also correlated with mucosal healing in ulcerative colitis patients treated with infliximab (de Bruyn et al., 2014, Inflamm. Bowel Dis., 20, 1198-1207).
The role of MMP9 has been associated with various neurological disorders, for example Alzheimer's disease (Mroczko et al., 2013, J. Alzheimers Dis., 37(2): 273-278), multiple sclerosis (Mirshafiey et al., 2014, Sultan Qaboos Univ Med J, 14(1), 13-25), neuroinflammation or cerebral ischemia (Candelario-Jalil et al., 2009, Neuroscience 158(3):983-94). In Alzheimer's disease the proaggregatory influence on tau oligomer formation in strategic brain regions may be a potential neurotoxic side effect of MMP9 (Wang et al., 2014 Bio Med Res. Int., 2014, ID 908636: 1-8). It has been suggested that a reduction in mature nerve growth factor (mNGF) as a consequence of elevated MMP9-mediated degradation, which degrades mNGF in the extracellular space, may in part underlie the pathogenesis of cognitive deficits in mild cognitive impairment and Alzheimer's disease (Bruno et al., 2009, J Neuropathol. Exp. Neurol., 68(12): 1309-1318).
The role of MMP9 has been associated with fibrotic diseases for example systemic sclerosis, multifocal fibrosclerosis, sclerodermatous graft versus host disease in bone marrow transplant recipients, nephrogenic systemic fibrosis, as well as organ-specific disorders such as pulmonary, liver, and kidney fibrosis (Piera-Velazquez et al., 2011, Am J Pathol., 179(3):1074-80, Peng et al., 2012, J. Clin. Immunol., 32(6):1409-14). For example recent studies have shown that MMPs, in particular MMP9, are implicated in initiation and progression of kidney fibrosis through tubular cell epithelial-mesenchymal transition and activation of resident fibroblasts, endothelial-mesenchymal transition and pericyte-myofibroblast transdifferentiation (Zhao et al., 2013, World J Nephrol., 2(3):84-9).
The pathophysiology of various eye diseases has been associated with MMP9 activity. Some examples include: fibrotic pathologies of the lens (Nathu et al., 2009, Ex. Eye Res., 88(2): 323-330), corneal diseases that is associated with up-regulation of MMP9 (Sakimoto et al., 2012, Cornea 31, Suppl 1:S50-6), diabetic retinopathy that presents increased MMP9 levels in patients retina and vitreous (Kowluru et al., 2012, Expert Opin Investig Drugs, 21(6): 797-805) and age-related macular degeneration where MMP9 was shown to play a role in its pathogenesis (Nita et al., 2014, Med Sci Monit, 20:1003-16).
Cardiovascular diseases involve inflammation and altered tissue remodeling associated with the reorganization of extracellular matrix and the activation of MMP9. Therefore, MMP9 is thought to be associated with pathophysiology of cardiac diseases such as hypertension, atherosclerosis, myocardial infarction, heart failure and coronary artery disease (Yabluchanskiy et al., 2013, Physiology, 28(6):391-403).
Furthermore, the role of MMP9 has been associated with various groups of disorders such as skin diseases (Mezentsev et al., 2014, Gene, 540(1):1-10), sepsis and acute inflammatory shock syndrome (Lorento et al., 2014, PLoS One 9(4):e94318; Qui et al. 2012, Comb Chem High Throughput Screen., 15(7):555-70), osteoarthritis (Bian et al., 2012, Front Biosci (Elite Ed). 4:74-100), chemotherapy-induced mucositis (Al-Dasooqi et al., 2009, Cancer Chemother Pharmacol., 64: 1-9), oral diseases (Al-Azri et al., 2013, Oral Diseases, 19: 347-359), osteosclerosis (Teti et al., 1999, J Bone Miner Res. 14(12):2107-17), endometriosis (Pitsos et al., 2009, Reprod Sci., 16(8):717-26) or Chagas disease (Geurts et al., 2012, Pharmacol Ther., 133(3):257-79).
Both monomeric and dimeric forms of MMP9 have been identified in a variety of normal and tumor cells (Goldberg et al., 1992, J. Biol. Chem., 267, 4583-4591) and in biological fluids and tissues, indicating that both forms are physiologically relevant. In addition to proteolysis, dimerization of MMP9 through the hemopexin domain appears necessary for MMP9 enhanced cell migration (Dufour et al., 2010, J. Biol. Chem., 285, 35944-35956) and study of the secretion patterns of MMP9 monomer and dimer in a variety of carcinoma, sarcoma, adenosarcoma and leukemia cell lines revealed that high MMP9 and especially dimer secretion levels correlated with the most aggressive cancer cell lines (Roomi et al., 2014, Int. J. Oncol. 44, 986-992). All together, these observations highlight the importance for an effective MMP9 neutralizing agent to efficiently inhibits all natural forms of MMP9 and more particularly MMP9 dimer and NGAL/MMP9 complex to treat very aggressive metastatic cancers.
Historically, strategies for MMP blockade have focused on the design of small molecule inhibitors that interact intimately with the catalytic site of the activated enzyme. To date, this approach has failed to translate into the expected clinical benefit partly due to dose-limiting toxicity and severe side effects such as musculoskeletal syndrome. As the architecture of the MMP9 catalytic site is highly conserved across the MMP family, this contra-indication may be attributable to a lack of MMP target selectivity at therapeutic doses.
Antibodies or antibody fragments are likely to interact with, and occlude a far larger portion of the MMP9 structure than active-site directed small molecules providing higher target inhibitory selectivity.
Some antibodies specific for MMP9 have been described in the prior art such as mouse AB0041 and humanized AB0045 (WO 2013/130078) as well as human 539A-M0240-B03 (US 2009/0311245), M0166-F10 (US 2009/0311245 US 2011/0135573), 539A-M0237-D02 (US 2009/0297449 and US 2011/0135573), mouse REGA-3G12 (Martens et al., 2007, Biochim. Biophys. Acta 1770, 178-186). Some of the antibodies of the prior art have been described as binding to both MMP9 and MMP2.
Therefore, there remains a need for the development of novel therapeutic agents which show a high affinity and specificity for MMP9 and exhibit a weak or limited affinity and/or specificity to other MMPs such as MMP2, show improved cross-reactivity to non-human MMP9 orthologs, and possess other additional properties such as reduced immunogenicity in humans and/or higher stability, which rend them particularly suitable for therapeutic applications in humans.